Characterization and gene expression analysis of the cir multi-gene family of Plasmodium chabaudi chabaudi (AS)

The pir genes comprise the largest multi-gene family in Plasmodium, with members found in P. vivax, P. knowlesi and the rodent malaria species. Despite comprising up to 5% of the parasite genome, little is known about the functions of the proteins encoded by pir genes. P. chabaudi causes chronic infection in mice, which may be due antigenic variation. In this model, pir genes are called cirs and may be involved in this mechanism allowing evasion of host immune responses. We have annotated the cir repertoire and performed detailed bioinformatic characterization of the encoded CIR proteins. Two major sub-families were identified: A and B, which display different amino acid motifs, and are thus predicted to have undergone functional divergence. The expression of all cirs was analyzed via RNA sequencing and microarray. Up to 40% of cir genes were expressed in the parasite population during infection, including members of both sub-families. Dominant cir transcripts could also be identified. Finally, specific cir genes were expressed at different time points during the blood stages of infection. Together our data characterizing the cir genes and their expression throughout the intra-erythrocytic cycle of development indicate that CIR proteins are likely to be important for parasite survival in the host. P. chabaudi AS is a highly synchronous parasite for which development in the blood follows its host’s circadian rhythm. Twelve time-points were then collected; one every two hours, to cover the entire 24 h cycle of blood stage development. At the peak of parasitaemia, one mouse was sacrificed at each time point and thin blood films were made and stained with Giemsa for optical microscopy. The pan-rodent microarray was designed using the OligoRankPick program as previously described: Liew, K et al.2010, Defining species specific genome differences in malaria parasites. BMC genomics 11,128. The RNA preparation, Cy-dye coupling to cDNA, hybridization and slide scanning were performed as described by Bozdech and colleagues Bozdech, Z et al 2003, The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 1, E5.

pir基因是疟原虫属（Plasmodium）中规模最大的多基因家族，其同源基因分布于间日疟原虫（P. vivax）、诺氏疟原虫（P. knowlesi）以及啮齿类疟疾虫株中。尽管pir基因家族占疟原虫基因组总量的比例可达5%，但目前学界对其编码蛋白的功能仍知之甚少。查氏疟原虫（P. chabaudi）可引发小鼠慢性感染，这一现象可能源于抗原变异。在该实验模型中，pir基因被命名为cir基因（cir），且可能参与了帮助疟原虫逃逸宿主免疫应答的致病机制。本研究对cir基因库进行了系统注释，并对其编码的CIR蛋白（CIR）开展了全面的生物信息学表征分析。研究共鉴定出两个主要的亚家族：A亚家族与B亚家族，二者具有独特的氨基酸基序，据此推测二者已发生功能分化。本研究通过RNA测序（RNA sequencing）与基因芯片（microarray）技术，对所有cir基因的表达情况进行了系统性分析。感染阶段的疟原虫种群中，多达40%的cir基因可被检测到表达，涵盖两个亚家族的所有成员。同时还可鉴定出优势表达的cir转录本。最后，部分特异性cir基因在感染的红细胞内期不同时间点呈现差异性表达。综上，本研究针对cir基因及其在红细胞内发育周期中的表达特征所获得的全部数据表明，CIR蛋白很可能在疟原虫宿主内存活过程中发挥关键作用。

查氏疟原虫AS株（P. chabaudi AS）是一种高度同步化的疟原虫，其在宿主体内的血液发育过程与宿主的昼夜节律保持高度一致。本研究共采集12个时间点的样本，每两小时采集一次，以覆盖红细胞内期发育完整的24小时周期。在寄生虫血症（parasitaemia）峰值期，每个时间点均处死1只小鼠，制备薄血膜并以吉姆萨染色（Giemsa）法进行染色，用于光学显微镜观察。本研究使用OligoRankPick程序（OligoRankPick）设计了泛啮齿类基因芯片，具体方法参照此前发表的文献：Liew K等，2010年，《定义疟疾虫株的物种特异性基因组差异》，BMC Genomics，11卷，128页。RNA制备、Cy染料（Cy-dye）与互补DNA（cDNA）的偶联、芯片杂交以及玻片扫描操作，均参照Bozdech及其团队的经典方法：Bozdech Z等，2003年，《恶性疟原虫红细胞内发育周期的转录组》，PLOS Biology，1卷，E5。